Methimazole adsorbant sample slide

ABSTRACT

A method for analyzing or detecting methimazole (“MTZ”) comprising contacting a sample suspected of containing MTZ with the dendrimer-stabilized silver nanoparticles and performing surface-enhanced Raman scattering (SERS). Graphene-dendrimer-stabilized silver nanoparticles (G-D-Ag).

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Continuation of U.S. application Ser. No.15/920,115, now allowed, having a filing date of Mar. 13, 2018, andclaims priority to U.S. Provisional Application No. 62/471,524, having afiling date of Mar. 15, 2017 which is incorporated herein by referencein its entirety.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR

Related technology is described by Saleh, T. A. et al. GrapheneDendrimer-stabilized silver nanoparticles for detection of methimazoleusing Surface-enhanced Raman scattering with computational assignment.Sci. Rep. 6, 32185; doi: 10.1038/srep32185 (2016) which was publishedAug. 30, 2016.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure relates generally tographene-dendrimer-stabilized silver nanoparticles (G-D-Ag) and polymersupported derivatives and sheets thereof.

Description of Related Art

Raman spectroscopy is based on the behavior of the inelasticallyscattered photons upon interaction with targeted molecules, and it hasbeen recently becoming an attractive tool for various applications. Themost challenging problem with Raman techniques is the nature of the weakscattering, which hinders its effective utilization, especially for lowdetection limit targets. The surface-enhanced Raman scattering (“SERS”)approach, however, could provide a promising strategy to solve thisproblem. Moreover, given the noticeable advances in instrumenttechnology, Raman spectroscopy has begun to compete withwell-established traditional analytical techniques in terms ofsensitivity and ease of use; Fleischmann M., Hendra P. J. & McQuilla A.J. Raman spectra of pyridine adsorbed at a silver electrode. ChemicalPhysics Letters, 26, 2, 163-166 (1974).

In SERS, the targeted molecules are adsorbed from an aqueous solutiononto nanoparticles that allow a charge transfer between analytemolecules and the particle surface, leading to an enhancement of theRaman signal. See Tian Z. Q., Ren, B. & Wu D. Y. Surface-enhanced Ramanscattering: from noble to transition metals and from rough surfaces toordered nanostructures. Journal of Physical Chemistry B, 106, 37,9463-9483 (2002), incorporated herein by reference in its entirety.Among the various commonly used types of materials to produce enhancedscattered Raman light are high-purity film-based substrates, whichinclude metals settled on planar surfaces such as glass, quartz, andsilicon wafers; or on nanoparticle-embedded surfaces such as silicabeads and polystyrene. See Vanduyne R. P., Hulteen J. C. & Treichel D.A. Atomic force microscopy and surface enhanced Raman Spectroscopy, Agisland film over polymer nanosphere surfaces supported on glass. Journalof Chemical Physics, 99, 3, 2101-2115 (1993); and Giesfeldt, K. S. etal. Studies of the optical properties of metal-pliable polymer compositematerials. Applied Spectroscopy, 57, 11, 1346-1352 (2003), eachincorporated herein by reference in their entirety. SERS films can alsobe tuned somewhat to appropriate localized surface plasmon resonances byaltering various parameters such as film thickness and deposition rate,with most thicknesses of metal being between 5-60 nm. See De Jesus, M.A., Giesfeldt, K. S. & Sepaniak, M. J. Factors Affecting the Sorption ofModel Environmental Pollutants onto Silver-Polydimethylsiloxanenanocomposite Raman Substrates. Applied Spectroscopy, 58, 10, 1157-1164(2004), incorporated herein by reference in its entirety. SERSsubstrates of colloidal silver or gold nanoparticles can consistentlyyield a large signal enhancement, explained by electromagnetic and/orchemical enhancement. See Reilly T. H., Corbman J. D. & Rowlen K. L.Vapor Deposition Method for Sensitivity Studies on EngineeredSurface-Enhanced Raman Scattering-Active Substrates. AnalyticalChemistry, 79, 13, 5078-5081 (2007), incorporated herein by reference inits entirety.

Recently, SERS has been reported as a promising technique forquantitative and qualitative identifications of various targets. SeeNovikov, S. and Khriachtchev, L. Surface-Enhanced Raman Scattering ofSilicon Nanocrystals in a Silica Film. Sci. Rep. 6, 27027; doi:10.1038/srep27027 (2016), incorporated herein by reference in itsentirety. It demonstrated the potential to impact the areas ofanalytical chemistry, biochemistry, forensics, environmental analysis,and trace analysis.

The SERS approach exhibits a number of advantages for use inlow-detection limit drug analysis when compared to other analyticaltechniques. Due to its ultra-sensitivity, SERS was used to detect traceorganic and inorganic analytes in different media. For example, someorganophosphorus compounds, such as methylparathiol and dimethoate, thatexist in pesticides were identified at the nanogram level. SeeSzymanski, H. A. Raman Spectroscopy: Theory and Practice. Plenum Press,Buffalo, N.Y., 1967, incorporated herein by reference in its entirety.Because water molecules scatter weakly in Raman experiments, it has madethe SERS approach an attractive choice to conduct usefulcharacterization of samples. See Creighton J. A., Blatchford C. G. &Albrecht M. G. Plasma resonance enhancement of Raman scattering bypyridine adsorbed on silver or gold sol particles of size comparable tothe excitation wavelength, Journal of the Chemical Society, FaradayTransactions, 75, 790-798 (1979); Powell, J. A. et al. Programmable SERSactive substrates for chemical and biosensing applications usingamorphous crystalline hybrid silicon nanomaterial. Sci. Rep. 6, 19663;doi: 10.1038/srep19663 (2016); and Dogan, I. et al. Analysis of temporalevolution of quantum dot surface chemistry by surface-enhanced Ramanscattering. Sci. Rep. 6, 29508; doi: 10.1038/srep29508 (2016), eachincorporated herein by reference in their entirety.

However, one of the most challenging tasks in developing an effectiveanalytical SERS based method is the fabrication of the right metalcolloid substrate, such as silver, that can exhibit a hotspot within thenanoparticles and subsequently achieve extremely high enhancement. SeeAroca, R. F., Alvarez-Puebla, R. A., Pieczonka, N., Sanchez-Cortez, S.and Garcia-Ramos, J. V. Surface-enhanced Raman scattering on colloidalnanostructures. Advances in Colloid and Interface Science, 116, 1-345-61 (2005), each incorporated herein by reference in its entirety.Since it is required to have more nanoparticles to hook the targetedmolecules, the use of a support to load the silver nanoparticles maycontrol the agglomeration that diminishes the enhancement in SERS.Dendrimers, which represent a new class of polymeric nanoscalecompounds, are promising candidates for SERS applications due to theirhomogeneous nature and unique tree-like structure. They have been foundto be useful in the health industry, and in pharmaceutical and materialsapplications. See Abbasi, E. et al. Dendrimers: synthesis, applications,and properties. Nanoscale Research Letters 9, 247-257 (2014),incorporated herein by reference in its entirety. In addition,dendrimers are considered as one of the most appropriate encapsulatingagents for the stabilization of metal nanoparticles (NPs), due to theirlarge size and the presence of a unique three-dimensional architectureof the dendrons that prevents leaching of the NPs during the course ofthe reaction. See Jiang Y., Gao Q. Heterogeneous Hydrogenation Catalysesover Recyclable Pd(0) Nanoparticle Catalysts Stabilized by PAMAM-SBA-15Organic-Inorganic Hybrid Composites. J. Am. Chem. Soc. 128, 716-717(2006), incorporated herein by reference in its entirety. Thepolyamidoamine dendrimers are considered the favored choice forpharmaceutical applications, due to their regular structure, large size,and chemical versatility. See Rajesh R., Kumar S. S., & Venkatesan R.Efficient degradation of azo dyes using Ag and Au nanoparticlesstabilized on graphene oxide functionalized with PAMAM dendrimers. NewJ. Chem. 8, 1551-1558 (2014), incorporated herein by reference in itsentirety.

Several analytical procedures have been reported for the determinationof a methimazole-based drug (also known as 1-methyl-2-mercaptoimidazoleand tapazole), which is considered as an antihormone drug widely used totreat hyperthyroidism. These methods include molecularly imprintedbiomimetic sensing, fluorescence, thin layer chromatography, coulometry,conductometry, and high-performance liquid chromatography withultraviolet detection. See Pan, M. et al. Molecularly imprintedbiomimetic QCM sensor involving a poly(amidoamine) dendrimer asafunctional monomer for the highly selective and sensitive determinationof methimazole, Sensors and Actuators B: Chemical, 207, 588-595 (2015);Farzampour L., Amjadi M., Sensitive turn-on fluorescence assay ofmethimazole based on the fluorescence resonance energy transfer betweenacridine orange and silver nanoparticles, Journal of Luminescence, 155,226-230 (2014); Aletrari M., Kanari P., Partassides D., Loizou E., Studyof the British Pharmacopeia method on methimazole (thiamazole) contentin carbimazole tablets, Journal of Pharmaceutical and BiomedicalAnalysis, 16, 785-792 (1998); Nikolic K., Velasevic K. Coulometricdetermination of methimazole, Pharmazie, 42, 698-700 (1987); Berka A.,Velasevic K., Nikolic K. Conductometric determination of methimazole,Pharmazie, 44, 499-500 (1989); and Moretti, G. et al. Determination ofthyreostatic residues in cattle plasma by high-performance liquidchromatography with ultraviolet detection, Journal of Chromatography:Biomedical Applications, 616, 291-296 (1993), each incorporated hereinby reference in their entirety. No SERS attempts with the use ofgraphene dendrimeric-based substrates have been reported to detectlow-concentration samples of methimazole (“MTZ”).

Other work in this field includes that of Liao X. et al. Au—Ag—Au doubleshell nanoparticles-based localized surface plasmon resonance andsurface-enhanced Raman scattering biosensor for sensitive detection of2-mercapto-1-methylimidazole. Talanta. 117, 203-208 (2013); Ma P. et al.Highly sensitive SERS probe for mercury(II) using cyclodextrin-protectedsilver nanoparticles functionalized with methimazole. Microchimica Acta.181, 975-98 (2014); Economou A., Tzanavaras P. D., Notou M. & ThemelisD. G. Determination of methimazole and carbimazole by flow-injectionwith chemiluminescence detection based on the inhibition of theCu(II)-catalysed luminolz-hydrogen peroxide reaction. Analytica ChimicaActa. 505, 129-133 (2004); Sun J. et al. Electrochemical Detection ofMethimazole by Capillary Electrophoresis at a Carbon Fiber MicrodiskElectrode. Electroanalysis. 17, 1675-1680 (2005); Yazhen W.Electrochemical determination of methimazole based on the acetyleneblack chitosan film electrode and its application to rat serum samples.Bioelectrochemistry. 81, 86-90 (2011); Zakrzewski R. Determination ofMethimazole in Pharmaceutical Preparations using an HPLC Method Coupledwith an Iodine-Azide Post-Column Reaction. Journal of LiquidChromatography & Related Technologies. 32, 383-398 (2009); and MoleroL., Faundez M., Valle M. A., del Rio R. & Armijo F. Electrochemistry ofmethimazole on fluorine-doped tin oxide electrodes and its square-wavevoltammetric determination in pharmaceutical formulations.Electrochimica Acta. 88, 871-876 (2013).

In this work the inventors used graphene as a support, modified with adendrimer, to allow controlled silver nanoparticles to be linked to itsbranches. The prepared graphene linked with dendrimer-stabilized silvernanoparticles (G-D-Ag) was used as a SERS substrate for MTZ detection.

BRIEF SUMMARY OF THE INVENTION

A method for analyzing or detecting methimazole (“MTZ”) comprisingcontacting a sample suspected of containing MTZ with thedendrimer-stabilized silver nanoparticles and performingsurface-enhanced Raman scattering (SERS). Graphene-dendrimer-stabilizedsilver nanoparticles (G-D-Ag) such as those comprising a graphene oxidesheet supported polyamidoamine (“PAMAM”) dendrimer represented byformula (I):

-   -   wherein X is -A-B—NH₂, -A-B—N-(A-B—NH₂)₂, or        -A-B—N-[A-B—N-(A-B—NH₂)₂]₂,        -   A is —CH₂CH₂C(O)—;        -   B is —NHCH₂CH₂—;        -   Graphene represents the graphene oxide sheet; and        -   m is a positive integer in the range of 2-100; and

wherein a weight ratio of the silver nanoparticles relative to thegraphene oxide sheet is in the range of 1:1 to 3:1.

Other advantages and features will become more clearly apparent from thefollowing description of particular embodiments of the invention, whichare given by way of non-limiting example and illustrated in the figureslisted below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Illustration explaining the synthesis steps of thegraphene-polyamidoamine dendrimer-silver G-D-Ag.

FIG. 2 : Mechanism of the stabilization of the AgNPs on the graphenethrough the dendrimer for the preparation of graphene-polyamidoaminedendrimer-silver (G-D-Ag).

FIG. 3 . The optimized structure of MTZ.

FIG. 4 . UV-Vis absorption spectra of (a) the G-D and (b) the G-D-Ag.

FIG. 5 . FT-IR spectra of (a) G-D and (b) G-D-Ag.

FIG. 6A. Typical SEM image of G-D.

FIG. 6B. Typical SEM image of G-D-Ag.

FIG. 6C. EDX spectra of G-D.

FIG. 6D. EDX spectra of G-D-Ag.

FIG. 6E. Mapping image of G-D.

FIG. 6F. Mapping image of G-D-Ag.

FIG. 6G. TEM image of G-D-Ag.

FIG. 7 . Raman spectra of (a) G-D and (b) G-D-Ag.

FIG. 8 . Raman spectrum of (a) pure solid MTZ and (b) SERS spectrum of1×10⁻⁵ M MTZ with G-D-Ag as a substrate, Laser λ=633 nm, acquisitiontime; 20 sec, and objective; 50×.

FIG. 9A. SERS spectra of MTZ with different concentration using G-D-Ag.Spectral plots appear in the order indicated with le-6 M being theuppermost plot and le-11 M being the lowermost.

FIG. 9B. Calibration curve of the band at 1359 cm⁻¹. Laser λ=633 nm,acquisition time; 20 sec, and objective; 50×.

FIG. 10 . Form 1 sample holder design for Raman measurements.

FIG. 11 . Form 2 sample holder design for Raman measurements.

DETAILED DESCRIPTION OF THE INVENTION

Hyperthyroidism (overactive thyroid) is a condition in which yourthyroid gland produces too much of the hormone thyroxine.Hyperthyroidism can accelerate your body's metabolism significantly,causing sudden weight loss, a rapid or irregular heartbeat, sweating,and nervousness or irritability. Several treatment options are availablefor hyperthyroidism. Anti-thyroid medications and radioactive iodine areused to slow the production of thyroid hormones. Sometimes, treatment ofhyperthyroidism involves surgery to remove all or part of the thyroidgland. Although hyperthyroidism can be serious if it is ignored mostpeople respond well once hyperthyroidism is diagnosed and treated.Hyperthyroidism is also found in animals. Clinical hyperthyroidism incats and dogs is produced from excessive secretion of the thyroidhormones, T₄ and T₃, resulting in signs that reflect an increasedmetabolic rate. It is most common in middle-aged to old cats and is lessfrequently seen in dogs.

Methimazole (1-methylimidazole-2-thiol) is a white, crystallinesubstance that is freely soluble in water. It differs chemically fromthe drugs of the thiouracil series primarily because it has a 5-insteadof a 6-membered ring. In some embodiments a methimazole derivative orprodrug may be detected, such as those described by U.S. Pat. No.6,365,616 B1 or by Roy, et al., J. Am. Chem. Soc., 2005, 127 (43), pp15207-15217 (both incorporated by reference).

Methimazole is readily absorbed in the gastrointestinal tract,metabolized in the liver, and excreted in the urine. Methimazoleprevents the thyroid gland from producing too much thyroid hormone. Itis used to treat hyperthyroidism but can cause side-effects such asagranulocytosis and liver inflammation.

Methimazole is contraindicated in the presence of hypersensitivity tothe drug or any of the other product components. Methimazole readilycrosses placental membranes and can cause fetal harm, particularly whenadministered in the first trimester of pregnancy and if methimazole isused, the lowest possible dose to control the maternal disease should begiven.

Agranulocytosis is a potentially a life-threatening adverse reaction ofMethimazole therapy. The drug should be discontinued in the presence ofagranulocytosis, aplastic anemia (pancytopenia), ANCA-positivevasculitis, hepatitis, or exfoliative dermatitis, and the patient's bonemarrow indices should be monitored. Although there have been reports ofhepatotoxicity (including acute liver failure) associated withMethimazole, the risk of hepatotoxicity appears to be less withMethimazole than with propylthiouracil, especially in the pediatricpopulation. Symptoms suggestive of hepatic dysfunction (anorexia,pruritus, right upper quadrant pain, etc.) should prompt evaluation ofliver function (bilirubin, alkaline phosphatase) and hepatocellularintegrity (ALT., AST). Drug treatment should be discontinued promptly inthe event of clinically significant evidence of liver abnormalityincluding hepatic transaminase values exceeding 3 times the upper limitof normal. Methimazole can cause hypothyroidism necessitating routinemonitoring of TSR and free T4 levels with adjustments in dosing tomaintain a euthyroid state. Because the drug readily crosses placentalmembranes, Methimazole can cause fetal goiter and cretinism whenadministered to a pregnant woman. For this reason, it is important thata sufficient, but not excessive, dose be given during pregnancy.

LFTs (liver function tests) are a group of blood tests that can help toshow how well a person's liver is working. LFTs include measurements ofalbumin, various liver enzymes (ALT, AST, GGT and ALP), bilirubin,prothrombin time, cholesterol and total protein.

Graphene is an allotrope of carbon in the form of a two-dimensional,atomic-scale, honey-comb lattice in which one atom forms each vertex. Itis the basic structural element of other allotropes, including graphite,charcoal, carbon nanotubes and fullerenes. Graphenes include bulkgraphite having more than ten graphene layers stacked, few-layergraphene (FLG): Two-dimensional material consisting of three to ten welldefined stacked graphene layers, bilayer graphene (2LG) two-dimensionalmaterial consisting of two well-defined stacked graphene layers; andmonolayer graphene (1LG) a single layer of carbon atoms with each atombound to three neighbors in a honeycomb structure. Carbon nanotubes,which have different structures and properties than most graphenes, maybe excluded.

Graphene oxides are depicted in FIGS. 1 and 2 . Acylated graphenescontain an acyl halide group, such as a —COX functional group, asdepicted by FIG. 1 , second diagram, which consists of a carbonyl groupsingly bonded to a halogen atom X. Halide anions include fluoride (F⁻),chloride (Cl⁻), bromide (Br⁻), iodide (I⁻) and astatide (At⁻) with acylchlorides and acyl iodides being preferred. A graphene functionalizedwith a dendrimer may be produced by reacting an acylated graphene (e.g.,graphene containing acyl halides) with ethylenediamine or anotherdiamine.

Dendrimers are repetitively branched molecules. Synonymous terms fordendrimer include arborols and cascade molecules. A dendrimer istypically symmetric around a core and often adopts a sphericalthree-dimensional morphology. In the invention dendrimers are attachedto a graphene, such as a graphene oxide surface. Dendrimers areclassified by generation, which refers to the number of repeatedbranching cycles that are performed during its synthesis. For example,if a dendrimer is made by convergent synthesis and the branchingreactions are performed onto the core molecule three times, theresulting dendrimer is considered a third generation dendrimer. Eachsuccessive generation results in a dendrimer roughly twice the molecularweight of the previous generation. Higher generation dendrimers, such asgenerations 4, 5, 6, 7, 8, 9, 10, 11 or 12 have more exposed functionalgroups on the surface which can later be used to customize the dendrimerfor a given application. These include polyamidoamine dendrimer which isdescribed in the Example.

Polyamidoamine (PAMAM) dendrimers are hyperbranched polymers withunparalleled molecular uniformity, narrow molecular weight distribution,defined size and shape characteristics and a multifunctional terminalsurface. These nanoscale polymers consist of an ethylenediamine core, arepetitive branching amidoamine internal structure and a primary amineterminal surface.

In some embodiments, the ethylene diamine moiety may be replaced byanother diamine that can be used to form a dendrimer, including diamineshaving 3-7 carbon atoms. These include 3 carbon diamines like1,2-diaminopropane or 1,3-diaminopropane, 4 carbon diamines such asputrescine (butane-1,4-diamine), 5 carbon diamines such as cadaverine(pentane-1,5-diamine) and 6 carbon diamines such as hexamethylenediamine(hexane-1,6-diamine).

Methyl acrylate is used in the Example to produce dendrimers. In otherembodiments, other acrylates may be used including acrylates containing5-21 carbon chain lengths and others described by Sabahi, et al.,Volume: 29 issue: 7, page(s): 941-953 (2014, incorporated by reference).

Dendrimers are “grown” off a central core in an iterative manufacturingprocess, with each subsequent step representing a new “generation” ofdendrimer. Increasing generations (molecular weight) produce largermolecular diameters, twice the number of reactive surface sites andapproximately double the molecular weight of the preceding generation.PAMAM dendrimers also assume a spheroidal, globular shape at Generation4 and. Their functionality is readily tailored, and their uniformity,size and highly reactive “molecular Velcro” surfaces are the functionalkeys to their use. Dendrimers such as PAMAM dendrimers appearing in theExample below are described and incorporated by references to thereferences cited herein.

Silver particles include the silver nanoparticles described in theExample and Figures. Dendrimer templated construction of silvernanoparticles is described by Castonguay, et al., Advances in Colloidand Interface Science, Volume 160, Issues 1-2, 15 Oct. 2010, Pages76-87; and encapsulation of silver nanoparticles into graphite graftedwith hyperbranched poly(amidoamine) dendrimer and their catalyticactivity towards reduction of nitro aromatics by Rajesh, et al., Journalof Molecular Catalysis A: Chemical, Volume 359, July 2012, Pages 88-96,both of which are incorporated by reference. The use of silver particleshooked the graphene surface support via dendrimers avoided problemsassociated with the use of naked silver particles such as agglomeration.

Those skilled in the art will select silver compounds and reducingagents suitable for decorating a dendrimer with silver nanoparticles asshown by FIG. 2 . In general, different reducing agents such as sodiumcitrate, ascorbate, sodium borohydride (NaBH₄), elemental hydrogen,polyol process, Tollens reagent, N, N-dimethylformamide (DMF), and poly(ethylene glycol)-block copolymers are used for reduction of silver ions(Ag⁺) in aqueous or non-aqueous solutions. In the Example, sodiumborohydride is used as a reducing agent to decorate or bind silvernanoparticles to a dendrimer. Formation of silver nanoparticles byreduction is also described by and incorporated by reference to Iravani,et al., Synthesis of silver nanoparticles: chemical, physical andbiological methods, Res Pharm Sci. 2014 November-December; 9(6):385-406.

In some embodiments only silver particles will be associated with ordecorated on the dendrimer component of the invention and metals such asAu, Cu, Fe, Ir, Ni, Os, Pd, Pt, and Ru and alloys (or metal compoundssuch as metal sulfides) thereof will not be present.

Nanoparticles or nanosized particles refer to particles having a meanparticle size ranging from 1 nm to ≤100 nm which range includes allintermediate values and subranges, such as 1, 2, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 75, <100 and 100 nm, for example, as determinedusing transmission electron microscopy (“TEM”). In some preferredembodiments silver nanoparticles will have mean diameters of less than35, 36, 37, 38, 39 or 40 nm. Nanoparticles according to the inventionadvantageously may have a mean diameter of less than 1, 2, 5, 10, 15,20, 25 or 50 nm and encompass particles that are 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, 100% or more smaller or larger thanthose described in the Example and Figures (or any intermediate value orsubrange of the ranges above).

Pharmaceutical products. Among its other uses, the method of theinvention can be applied to detect methimazole in a pharmaceuticalcomposition. The invention may be used to assess product purity, detectcounterfeit drugs, detect batch-to-batch differences in methimazolepreparations, detect or monitor degradation of methimazole over time orof methimazole stored under different temperatures or conditions, ordetect spatial or lateral distribution of methimazole in a tablet,granule or other pharmaceutical preparation, assess particle size of aparticulate pharmaceutical preparation, or to help explaininconsistencies in dissolution profiles of methimazole.

Biological samples include samples from both in vivo and in vitrosources, such as samples taken from a patient taking methimazole or fromcells exposed to methimazole. Biological samples include blood, plasma,serum, and urine or other samples suspected of containing methimazole.

Environmental, Industrial, Commercial or other samples. The methoddescribed herein may detect methimazole in virtually any form, includingin forensic samples, environmental samples, or industrial samples.

Detection sensitivity. In some embodiments, the detection limit of amethod of the invention will be at least 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M(or any intermediate value within this range). As shown in the Examplebelow, a low detection limit of 1.43×10⁻¹² M was successfully obtained.

Selected embodiments of the invention include, but are not limited tothose described below.

Embodiment 1. A Graphene-dendrimer-stabilized silver nanoparticles(G-D-Ag) that comprises a graphene oxide sheet supported polyamidoamine(“PAMAM”) dendrimer represented by formula (I):

-   -   wherein X is -A-B—NH₂, -A-B—N-(A-B—NH₂)₂, or        -A-B—N-[A-B—N-(A-B—NH₂)₂]₂,        -   A is —CH₂CH₂C(O)—;        -   B is —NHCH₂CH₂—;        -   Graphene represents the graphene oxide sheet;

wherein m is a positive integer in the range of 2-100 (or anyintermediate integer value or subrange such as 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100); and

silver nanoparticles bound to the graphene oxide sheet supportedpolyamidoamine (“PAMAM”) dendrimer;

wherein a weight ratio of the silver nanoparticles relative to thegraphene oxide sheet is in the range of 1:1 to 3:1 (or any intermediatevalue or subrange such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6,1:2.7, 1:2.8, 1:2.9 or 1:3.

Embodiment 2. The G-D-Ag of embodiment 1, wherein the silvernanoparticles have a mean diameter of no more than 40 nm, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25,30, 32, 35, 36, 37, 38, 39, <40 or 40 nm (or any intermediate value orsubrange).

Embodiment 3. The G-D-Ag of embodiment 1, wherein the silvernanoparticles have a mean diameter of no more than 20 nm.

Embodiment 4. A SERS-active material comprising the G-D-Ag of embodiment1 and a Surface-Enhanced Raman Scattering (SERS) active substrate thatcomprises silica glass coated with at least one layer of the G-D-Ag.

Embodiment 5. A SERS-active material comprising the G-D-Ag of embodiment1 and a Surface-Enhanced Raman Scattering (SERS) active substrate thatcomprises silica glass coated with at least one layer of the G-D-Ag,wherein said at least one layer of G-D-Ag in aggregate ranges inthickness from 10 nm to 100 μm, such as 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or <1,000 nm, or suchas 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, <100, or 100 μm (or anyintermediate value or subrange).

Embodiment 6. A method for detecting methimazole (“MTZ”) or determininga concentration of methimazole in a sample comprising contacting thesample containing or suspected of containing MTZ with thedendrimer-stabilized silver nanoparticles of embodiment 1 and performingsurface-enhanced Raman scattering (SERS).

Embodiment 7. The method of embodiment 6 for determining a concentrationof methimazole in at least one sample, comprising:

mixing the at least one sample with G-D-Ag at a volume ratio of 1:1 to8:1 to prepare at least one analyte; and performing surface-enhancedRaman scattering (“SERS”) by acquiring a SERS spectrum of the at leastone analyte by Raman spectroscopy;

determining the concentration of methimazole in the at least one sampleby comparing the peak intensity of a Raman band of methimazole obtainedfrom the SERS spectrum of the at least one analyte to a standard linearregression curve that plots known concentrations of methimazole againstpeak intensities of the Raman band.

Embodiment 8. The method of embodiment 6, wherein the sample is apharmaceutical, drug or chemical sample and not a biological sample froma subject.

Embodiment 9. The method of embodiment 6, further comprising determiningpurity of a nonbiological pharmaceutical sample of methimazole, whetherthe sample is counterfeit, whether there is a difference between two ormore methimazole samples, whether a methimazole sample has degraded,spatial or lateral distribution of methimazole in a sample that is atablet or granule, or determining a particle size of a pharmaceuticalpreparation containing methimazole. Peak intensities from differentsamples may be compared to those of control samples of known purity,those of methimazole stored for a particular period of time or underparticular temperature, humidity or other physical or chemicalconditions, or to those of samples having particular particle sizes orspatial distributions of methimazole.

Embodiment 10. The method of embodiment 6, wherein the sample is serum,plasma, urine or other biological sample.

Embodiment 11. The method of embodiment 6, wherein the sample is humanserum, plasma, urine or other human biological sample.

Embodiment 12. The method of embodiment 6, wherein the sample is serum,plasma, urine or other biological sample obtained from Felis catus(domestic cat) or other member of the family Felidae.

Embodiment 13. The method of embodiment 6, wherein the sample is from asubject having hyperthyroidism or at risk thereof.

Embodiment 14. The method of embodiment 6, wherein the sample is from afemale who is pregnant or who may become pregnant.

Embodiment 15. The method of embodiment 6, wherein the sample is from asubject having, genetically predisposed to having, or at risk of havingagranulocytosis, aplastic anemia (pancytopenia), ANCA-positivevasculitis, exfoliative dermatitis, hepatitis, or hepatatic dysfunctionor at least one symptom thereof; or wherein the sample is from a subjecthaving anorexia, pruritus, or right upper quadrant pain or other symptomof hepatic dysfunction or wherein the sample is from a subject having anabnormal liver function test.

Embodiment 16. The method of embodiment 6, further comprising detectingat least one other analyte besides methimazole.

Embodiment 17. A method of synthesizing the G-D-Ag of embodiment 1,comprising: reacting an acylated graphene with ethylenediamine to form adendrimer G0; successively reacting the dendrimer G0 with methylacrylate followed by ethylenendiamine once to form a graphene sheetsupported PAMAM dendrimer G1, wherein X is -A-B—NH₂, twice to form agraphene sheet supported PAMAM dendrimer G2, wherein X is-A-B—N-(A-B—NH₂)₂, or three times to form a graphene sheet supportedPAMAM dendrimer G3, wherein X is -A-B—N-[A-B—N-(A-B—NH₂)₂]₂; andreacting the graphene sheet supported PAMAM dendrimer G1, G2, or G3 witha silver(I) salt in the presence of a reducing agent to form the G-D-Ag.

Embodiment 18. A system for analyzing a pharmaceutical preparation ofmethimazole or a biological sample containing methimazole comprising thedendrimer-stabilized silver nanoparticles (G-D-Ag), Raman spectroscope(e.g., excitation source, sampling apparatus, and detector),communications elements, data processing elements, software or computerequipment for analyzing, processing and storing these data, displays orother data output elements, and/or instructions for use in analyzingMTZ.

Embodiment 19. A kit for detecting methimazole in a pharmaceutical orbiological sample comprising the dendrimer-stabilized silvernanoparticles (G-D-Ag), and optionally one or more reagents suitable fordetecting MTZ in conjunction with G-D-Ag, one or more positive controlsamples, one or more negative control samples, one or more containers orreaction vessels, packaging materials and/or instructions for use indetecting MTZ, or promotional materials.

A sample holder comprising the Graphene-dendrimer-stabilized silvernanoparticles (G-D-Ag) of claim 1 and (a) a cuvette with dimensions of0.5 to 2.0 cm in diameter and 0.3 to 2.0 cm in length containing orcoated with the G-D-Ag, or (b) a silica glass slide having withdimensions of 3 to 10 cm in length and 1 to 5 cm in width that is coatedon at least one side with the G-D-Ag. Thickness of a coating or layer ofG-D-Ag may range from 1, 2, 5, 10, 20, 50, 100, 500, 1,000 nm to >1, 2,5, 10, 20, 50, 100 or >100 μM or any intermediate value or subrange.

EXAMPLE

As shown herein graphene functionalized with polyamidoamine dendrimer,decorated with silver nanoparticles (G-D-Ag), was synthesized andevaluated as a substrate with surface enhanced Raman scattering (SERS)for methimazole (MTZ) detection. Sodium borohydride was used as areducing agent to cultivate silver nanoparticles on the dendrimer. Theobtained G-D-Ag was characterized by using UV-vis spectroscopy, scanningelectron microscope (SEM), high-resolution transmission electronmicroscope (TEM), Fourier-transformed infrared (FT-IR) and Ramanspectroscopy. The SEM image indicated the successful formation of theG-D-Ag. The behavior of MTZ on the G-D-Ag as a reliable and robustsubstrate was investigated by SERS, which indicated mostly a chemicalinteraction between G-D-Ag and MTZ. The bands of the MTZ normal spectraat 1538, 1463, 1342, 1278, 1156, 1092, 1016, 600, 525 and 410 cm⁻¹ wereenhanced due to the SERS effect. Correlations between the logarithmicalscale of MTZ concentrations and SERS signal intensities were establishedand a low detection limit of 1.43×10⁻¹² M was successfully obtained. Thedensity functional theory (DFT) approach was utilized to providereliable assignment of the key Raman bands.

Experimental Procedure

Chemicals and Materials. Methimazole (MTZ) “1-Methyl-2-imidazolethiol”(analytical standard, ≥99% purity), CAS number 60560, was purchased fromSigma-Aldrich. Silver nitrate (AgNO₃, 99.8%), product number 30087, waspurchased from BDH-Chemicals Ltd Poole England. Sodium borohydride(NaBH₄), product number 63390, was purchased from Allied Signal.Ethylenediamine (≥99.5%), product number 03550, methyl acrylate (99%),CAS number 76778, thionyl chloride (SOCl₂, ≥99%), product number 230464,and potassium bromide (KBr, ≥99%), product number 221864, were purchasedfrom Sigma-Aldrich. Solutions were prepared with ultrapure waterobtained from a water purification system (Ultra Clear™ Lab WaterSystems, Siemens Water Technologies USA).

Synthesis of graphene dendrimer silver composite. FIG. 1 shows thepreparation steps of dendrimer functionalization with silver. About 0.2g of the prepared graphene nanosheets was dispersed in 20 ml of SOCl₂ bysonication in an ultrasound bath for 30 min and stirred for 12 h at 60°C.; the mixture was then filtered. The obtained material was driedovernight at room temperature. Next, 10 ml of ethylenediamine was addedto the solid product, the reaction mixture was sonicated for 3 h at 60°C. and stirred for another 12 h at room temperature. The solid productwas collected by centrifugation at 10,000 rpm/min for 10 min and driedovernight at room temperature.

The last solid product was suspended in 10 ml methanol and was addeddropwise to 25 ml of 1:4 methyl acrylate-methanol solution understirring. The reaction mixture was treated in an ultrasonic bath at 60°C. for 2 hours and stirred for another 12 h at room temperature.

The solid product was collected by centrifugation at 10,000 rpm/min for10 min and dried overnight at room temperature. Afterward, the obtainedmaterial was immersed in 10 ml methanol, and then a 1:1 mixture of 10 mlof ethylenediamine-methanol was added at a rate 1 drop/sec to thesolution. The solution was placed in an ultrasonic bath at 50° C. for 5h and stirred for another 10 h at room temperature. The solid productwas collected by centrifugation and dried overnight at room temperature.The steps were repeated for methyl acrylate and ethylenediamine untilreaching the third-generation. The third-generation polyamidoaminedendrimer on the graphene (G-D) presented a typical morphology whencompared to the others obtained using higher dendrimer concentrations.

The solid of this material was dispersed in 20 ml de-ionized water bysonication in an ultrasound bath for 10 min. Then, 10 ml of 0.2 M AgNO₃was added dropwise with the previously dispersed solid and the mixturewas stirred for 1 hour. Then, 10 ml of a freshly prepared solution ofNaBH₄ was added to the solution and the solution was kept under stirringfor another 5 h. Finally, the mixture was filtered, and the obtainedmaterial was washed with deionized water several times. The greenishyellow isolated solid was dried overnight at room temperature. Thestabilization mechanism of the silver nanoparticles (AgNPs) on thegraphene nanosheets through the dendrimers is shown in FIG. 2 . Theabbreviation used for graphene modified with a third-generationpolyamidoamine dendrimer is G-D, while for graphene-dendrimer-silvernanoparticles it is G-G-Ag.

Material Characterization. Scanning Electron Microscope, JSM-6610LV,JEOL at 20 kV acceleration voltage equipped with energy-dispersive X-rayspectroscope, Mapping and transmission electron microscope (TEM, FEITecnai TF20) were employed to investigate the morphological andmicrostructural attributes of the synthesized material. The UV-Visiblespectra of the graphene and G-D-Ag were recorded on a genesis 10S UV-Visspectrophotometer (Thermo Scientific), using standard quartz cuvette atroom temperature between 250-650 nm. The samples were prepared bydilution the stock solution 4× with distilled water. FT-IR spectra ofsamples were recorded using a Perkin-Elmer IR spectrophotometer usingpotassium bromide (KBr) pellets, the pellet was designed by blending thesample and KBr with a ratio of 1:100. The FT-IR measurement was scannedat a range from 400 to 4000 cm⁻¹. The He—Ne laser source operating at0.5 W was utilized for sample excitation.

Sample Holder Design. The sample holders were designed in two shapes orforms. First design was a holder in for of cuvette with dimensions of0.5 to 2 cm in diameter and 0.3 to 2 cm in length, as shown in Scheme 1(FIG. 10 ). The second form is the design of slides where the preparednanomaterials were coated on the surface of the silica glass slides withdimensions of 3 to 10 cm in length and 1 to 5 cm in width, Scheme 2(FIG. 11 ). The thickness of the layer of the coated materials was inthe nano to micro meter size.

Surface-Enhanced Raman Scattering (“SERS”) spectroscopy. The SERSspectra of samples were obtained by using a Raman spectroscopy system—aLab Ram HR Evolution Raman spectrometer—equipped with an internal He—Ne17 mW laser at a 633 nm excitation wavelength. SERS samples wereprepared in a small cuvette by using a 4:1 volume ratio of aqueous MTZsolution to G-D-Ag. A 50× objective was used for focusing the laser beamto the solution. The data acquisition time was 20 sec with oneaccumulation for collection with each SERS spectra. A cuvette withdimensions of 1 cm radius and 2 cm height was used as a sample cell forthe Raman spectra. The SERS spectra were obtained in the range from400-2000 cm⁻¹.

Theoretical Calculations. Density functional theory (DFT) calculationswere employed to optimize the structure of MTZ and calculate itsvibrational frequencies at the ground level. The Gaussian 09 program wasused to carry out the DFT-B3LYP/6-311++G(d,p) level of calculation. SeeGaussian 09, Revision D.01, Frisch M. J., et al., Gaussian, Inc.,Wallingford Conn., (2013), incorporated herein by reference in itsentirety. Atomic displacements associated with each vibrational modewere carefully inspected using Gauss-View software and correspondingpotential energy distributions (PEDs) were computed with Vida softwarein order to provide reliable assignments of the normal Raman, as well asSERS spectra, of MTZ. See GaussView, Version 5.0, R. Dennington II, T.Keith, J. Millam, Semichem Inc., Shawnee Mission, K S, 2009; and JamrózM. H. Vibrational Energy Distribution Analysis: VEDA 4, program, Warsaw,2004-2010, each incorporated herein by reference in its entirety. Theminimum-energy structure of MTZ with atom numbering adopted is shown inFIG. 3 . The vibrational frequencies were compared to the solid stateRaman spectra (Table 1).

Structural Analysis of G-D and G-D-Ag. The ultraviolet-visible spectraof G-D and G-D-Ag are shown in FIG. 4 . The maximum absorption band at300 nm is attributed to the n-π* electronic transitions of thedendrimer. Moreover, the maximum absorption peak of G-D-Ag is at 400 nm,due to the plasmon resonance of G-D-Ag, indicating the formation AgNPson the surface of the dendrimer.

FT-IR was employed to confirm the chemical structure of G-D and G-D-Ag.FIG. 5 shows the FT-IR spectra of G-D and G-D-Ag. The FT-IR spectrum ofG-D shows a weak broadband at ˜3418 cm⁻¹, corresponding to the vibrationof NH₂. The very low-intensity peaks at 2923 cm⁻¹ and at 2854 cm⁻¹ areassigned to the symmetric and antisymmetric stretching vibrations ofCH2, respectively. The bands at 1654 and 1324 cm⁻¹ are assigned to C═Cand C═O, respectively. The FT-IR spectrum of G-D-Ag differs from that ofG-D, as evidenced by the weakening of the NH₂ band in the range 3350 to3450 cm⁻¹. It suggests that the AgNPs are stabilized in the G-D networkthrough this functional group. See Shen, J. et al. One Step Synthesis ofGraphene Oxide-Magnetic Nanoparticle Composite. J. Phys. Chem. C, 114,1498-1503 (2010), incorporated herein by reference in its entirety. Thedisappearance of the peak, attributed to C—O at 1324 cm⁻¹ in the G-D-Agspectrum, is probably due to the reduction of the oxygenated functionalgroups through the heat treatment process. See Rajesh et al.

SEM, EDX and mapping imagings were used as techniques complementary toTEM to investigate the appearance of the synthesized materials, as seenin FIG. 6 . The SEM images (FIG. 6A), shows the morphology of theprepared G-D, and the inset TEM image illustrates the formation ofmulti-dots of dendrimers on the graphene nanosheets. These dots are usedas bases, or cores, for attracting and catching the silver ions. Thepresence of reactive amine groups on the surface of dendrimer-modifiedgraphene was profited to allow e multipoint attachment of the AgNPsthrough the formation of linkages, (as shown in the mechanism FIG. 2 )which were further transformed to stable secondary amino linkages byreductive treatment with NaBH₄. This allows for the controlled growth ofAgNPs, as shown in the TEM image (FIG. 6G) and the SEM image, with TEMinset (FIG. 6B), which provide evidence that the Ag nanoparticles arewell dispersed as a consequence of the stabilization of the growingsilver by the different amide groups of the dendrimer. The nanoparticlescould be stabilized by interaction with the primary amino groupsremaining at the outer surface of the dendrimer. The mapping images,FIGS. 6E and 6F, indicate that the stabilized AgNPs were mostly uniformdispersed. Further characterization was confirmed by EDX spectra (FIGS.6C and 6D), which confirms the presence of the silver, with stronginteraction with the dendrimer, even after washing the sample severaltimes, followed by drying. Therefore, the graphene was successfully usedas an indirect support or the silver nanoparticles. The silvernanoparticles were decorated on the dendrimer branches rather than beingdirectly attached to the graphene. This material provides the best SERSenhancement for MTZ compared with the AgNPs loaded graphene, because thedendrimer allows better distribution of AgNPs on the nanosheets, asshown in the TEM image. Therefore, the role of the graphene was as asupport; however, the silver nanoparticles were located on the dendrimerbranches (linkers) rather directly attached on the graphene. This waythe silver nanoparticles were better distributed and decorated on thegraphene sheets surface as shown in the TEM image.

Raman Analysis of G-D and G-D-Ag. The Raman spectra of the G-D andG-D-Ag are shown in FIG. 7 . The Raman spectra of all samples displayedtwo prominent bands. While the D band around 1350 cm⁻¹ is associatedwith disordered sp3 carbon atoms, the G band around 1590 cm⁻¹corresponds to ordered sp2-hybridized carbon atoms. See Sarkar, S.,Bekyarova, E., Niyogi, S., Haddon, R. C. Diels-Alder Chemistry ofGraphite and Graphene: Graphene as Diene and Dienophile. J. Am. Chem.Soc. 133, 3324-3327 (2011), incorporated herein by reference in itsentirety. Further, the intensity ratio of D and G bands (ID/IG)increases. The ID/IG is used to assess the sp2/sp3 carbon ratio, whichrepresents the degree of disorder and the average size of the sp2 carbonatoms domains. The ratio for G-D-Ag, 1.56, was larger than that for G-D,1.22, suggesting that more graphitic domains are formed and the sp2cluster number is increased after introducing the silver via thereduction process. This reflects the functionalization of the AgNPs onthe dendrimer-modified graphene. See Fang M., Wang K., Lu H., Yang Y. &Nutt S. Covalent polymer functionalization of graphene nanosheets andmechanical properties of composites. J. Mater. Chem. 19, 7098-.7105(2009), incorporated herein by reference in its entirety. This can beexplained by the removal of some oxygen-containing functional groupsduring the reduction process, leading to the formation of high-levelfragmentation along the reactive sites of graphene dendrimer. SeeLin-jun, H. et al. Preparation of Graphene Silver Nanohybrid Compositewith Good Surface-Enhanced Raman Scattering Characteristics. Int. J.Electrochem. Sci., 11 398-405 (2016), incorporated herein by referencein its entirety.

Surface-Enhanced Raman Scattering (SERS) spectra of MTZ with G-D-Ag. Thecollected Raman spectrum for solid MTZ, compared with a 1×10⁻⁵ Mconcentration MTZ-(G-D-Ag) SERS spectrum, is depicted in FIG. 8 . Inorder to understand the nature of the interaction between the boundingof the MTZ molecules and the surface of the AgNPs, it is useful topropose proper band assignments for the normal Raman and SERS spectra.For reliable assignments, we conducted DFT assessments of thevibrational frequencies of the single MTZ molecule and compared themwith the corresponding ones resulting from the interaction between thesilver and MTZ. All these data are listed in Table 1.

The DFT method based on the hybrid B3LYP functional and split-valence6-311++G(d,p) basis set showed good agreement with the experimentalresults. The band observed at 1342 cm⁻¹ and at 1345 cm⁻¹ in the solidand solution Raman spectra, respectively, shifted to 1359 cm-1 in theSERS spectrum. This band shows the highest enhancement factor. The DFTcalculation attributes this band mostly to the N2-C4 stretching (withsome contribution from the ring and C6-N3-H bending) and successfullypredicts its slight shift to the lower frequency side. Moreover, themodes observed at 1538 and 1463 cm⁻¹ have shifted to 1522 and 1452 cm⁻¹,respectively, in the SERS spectrum with significant enhancement. PEDanalysis shows that these bands are associated with S—C and C—Nstretching modes (Table 1). The bands at 1278, 1156, 1092, 1016, and 600cm¹ in the normal Raman spectrum are shifted to 1320, 1141, 1090, 1037,and 619 cm⁻¹, respectively in the SERS spectrum. These bands show higherintensities in the SERS spectrum.

311++G(d,p) basis set showed good agreement with the experimentalresults. The band observed at 1342 cm⁻¹ and at 1345 cm⁻¹ in the solidand solution Raman spectra, respectively, shifted to 1359 cm-1 in theSERS spectrum. This band shows the highest enhancement factor. The DFTcalculation attributes this band mostly to the N2-C4 stretching (withsome contribution from the ring and C6-N3-H bending) and successfullypredicts its slight shift to the lower frequency side. Moreover, themodes observed at 1538 and 1463 cm⁻¹ have shifted to 1522 and 1452 cm⁻¹,respectively, in the SERS spectrum with significant enhancement. PEDanalysis shows that these bands are associated with S—C and C—Nstretching modes (Table 1). The bands at 1278, 1156, 1092, 1016, and 600cm⁻¹ in the normal Raman spectrum are shifted to 1320, 1141, 1090, 1037,and 619 cm⁻¹, respectively in the SERS spectrum. These bands show higherintensities in the SERS spectrum.

TABLE 1 Infrared, Raman, SERS and calculated DFT vibrational frequencies(cm⁻¹) of MTZ. Obs. Raman Raman Calc. Assignments with IR (Solid)(Solution) SERS MTZ MTZ-Ag Corresponding PEDs (%) 3531 3366 100% v(N3—H) 3159 w 3161 w 3166 m 3162 3166 97% v (C7—H) 3104 w 3105 w 3106 vw3142 3147 98% v (C6—H) 3012 w 3022 3021 95% v (C5—H11) 2999 2995 100% v(C5—H12) 2949 vw 2950 m 2960 m 2945 m 2936 2932 96% v (C5—H13) 1578 vs1579 s 1580 m 1567 w 1588 1581 63% v (C6═C7), 10% δ (N3—H) bend 1538 vw1522 vs 1509 1496 24% v (N2—C4), 15% v (C-C), 38% δ (H11—CH—12) bend1473 1467 23% v (S—C4), 14% v (C4—N) bend, 10% δ (N3—H) bend, 1479 vw1480 vs 1466 1457 72% δ CH_(Me) scissoring 1462 s 1463 vs 1460 vw 1452 s1459 1452 23% v (S—C4), 14% v (N3—C4), 12% δ (C—H)bend, 1403 m 1410 m1410 vw 1408 w 1415 1411 14% v (N2—C4), 14% v (N3—C6), 13% v (S—C4), 30%δ (C—H)bend 1339 vs 1342 s 1345 s 1359 vs 1315 1328 32% v (N2—C4), 11% δring bend, 19% δ C6—N3—H bend 1274 s 1278 rn 1281 m 1320 s 1285 1309 15%v (N2—C5), 19% δ N3— H(C6—H)bend, 14% δ ring breathing 1248 m 1252 vs1255 vw 1277 vw 1212 1237 51% v (N3—C4), 18% δ N3— H(C6—H)bend, 13% δ(C7—H)bend 1152 vs 1156 vs 1153 m 1141 m 1159 1150 16% v (N3—C6), 16% v(S—C4), 15% δ (H11—C—H12) rock, 1086 vw 1092m 1088 vw 1090 m 1089 109146% v (N3—C6), 14% δ (N3—H) bend, 21% δ (C7—H)bend 1014 s 1016 m 1017 vw1037 m 1013 1022 15% ring CH bend, 13% δ CHMe bend, 41% δ ring bend, 913 m  915 vs  916 s  937 w 913 923 12% v (N2—C4), 12% δ N3— H(C6—H)bend, 62% δ ring bend  818 w  810 vw  830 vw 806 818 89% γ (H—C6—C7—H)twist  673 vs  679 vw  684 vs  687 w 685 699 25% δ (C7—N2—C5) bend, 15%δ (C4—N2—C5) bend  643 vw  670 vw 650 667 47% ring CH bend, 39% γ(N3—C4—N2)  599 vw  600 vw  602 vw  619 m 603 623 78% γ CN ring bend. 527 vs  525 m  522 w  498 s 534 520 53% δ (S—C4—N3) bend, 25% δ(S—C4—N2),  493 vw 503 569 84% γ (N3—C6—C7)  411 s  410 s  410 m  427 m411 421 71% δ (S—C4—N2)  264 m  260 m  279 w 238 251 85% γ (C4—S) wag 208 vw  209 vw 207 220 76% γ ring

SERS Enhancement Factors of MTZ. The SERS enhancement factors (EFs) forthe vibrations of MTZ (1×10⁻³ M) on G-D-Ag to the corresponding bandobtained from 1.0 M saturated solution were calculated using thefollowing equation:EFs(δ SERS×C normal)/(δ normal×C SERS);

where δ and C are the Raman mode intensity and sample concentrations,respectively. The EFs for the SERS peaks of MTZ on G-D-Ag are given inTable 2. The EFs are not the same for the different MTZ modes; themaximum enhancement was observed at 1342 cm⁻¹

TABLE 2 SERS enhancement factor of MTZ on G-D-Ag substrate. SERS spectra(cm⁻¹) Enhancement Factor (EF) 1522 8.3 × 10⁴ 1452 1.1 × 10⁴ 1359 1.5 ×10⁴ 1320 2.5 × 10⁴ 1141 1.0 × 10⁴ 1090 2.3 × 10⁴ 1037 3.8 × 10⁴ 619 1.4× 10⁴ 498 2.0 × 10⁴ 427 2.4 × 10⁴

SERS Spectra of MTZ at Different Concentrations. The SERS spectra of MTZaqueous solution with G-D-Ag as a substrate at different concentrationsare given in FIG. 9A. The intensities of the SERS spectra increase withan increase in the concentration of MTZ. This suggests that the SERSintensities are proportional to the molecular quantity of MTZ. Thehighest enhanced band, at 1359 cm⁻¹ in the SERS spectra, was selectedfor creating a qualitative analysis of MTZ. A plot of the ERS responseversus the logarithmical scale of 10⁻⁶ M to 10⁻¹¹ M of MTZ at 1359 cmwas obtained, (FIG. 9B), showing a good coefficient of determination(R²) of 0.9976. Within the dynamic range, the lowest concentrationmeasured in the SERS analysis of the MTZ solution was 10⁻¹¹ M. Toevaluate the analytical performance of the proposed method, parameterssuch as linearity, repeatability, limits of detection and dynamic rangewere investigated under optimum experimental conditions. The results ofthe linear equations, dynamic range, and R2 for the obtained calibrationcurves of MTZ with G-D-Ag substrate are summarized in Table 3.

TABLE 3 Regression equation between Raman intensities and concentrationsof MTZ and their coefficient of determination (R²). Dynamic linear RamanPeaks Regression Equation R² range (M) LOD* (M) 1359 cm⁻¹ y = 292.43x +3409.8 0.9976 10⁻⁶-10⁻¹¹ 1.43 × 10⁻¹² 1320 cm⁻¹ y = 144.97x + 1651.90.9921 10⁻⁶-10⁻¹¹ 2.67 × 10⁻¹²  498 cm⁻¹ y = 124.14x + 1479 0.974410⁻⁶-10⁻¹¹ 3.71 × 10⁻¹²  427 cm⁻¹ y = 63.771x + 739.39 0.9651 10⁻⁶-10⁻¹¹0.91 × 10⁻¹²

Good linear relations between the enhanced SERS bands' intensities andthe logarithmical scale of MTZ concentrations were noted with a widedynamic linear range or linear working range (LWR) for MTZ with thesubstrate. The precision of the proposed method was checked by replicateanalysis of the working standard of MTZ drug at six concentrationlevels. The relative standard deviation (RSD) for all concentrationlevels was <2.2%, which indicates both the precision and repeatabilityof the proposed method. The reproducibility of the method using the samebatch of the prepared material was obtained in five days, with acorresponding relative average standard deviation of less than 4%.

The results obtained by the reported method in this study were comparedwith some methods reported in the literature in terms of calibrationrange, detection limits, and determination coefficients (R²). Thecomparison with other methods for the determination of MTZ is summarizedin Table 4. In comparison to other methods for determination of the MTZ,the proposed method has attracted more interest due to its sensitivity,good dynamic range, and simplicity.

TABLE 4 Comparison of Dynamic linear range, detection limits between andcoefficient of determination (R²) this method and other methods for thedetermination of MTZ. Dynamic linear range Limit of detection Method (M)(M) R² Ref. SERS  10⁻⁶-10⁻¹¹ See Table 3 See Table 3 Present work SERS5.0 × 10⁻⁸-5.5 × 10⁻⁷   7.4 × 10-05 0.998 29 SERS 1.8 × 10⁻⁹-1.3 × 10⁻⁶  8.8 × 10⁻¹⁰ 0.9992 30 Flow-Injection 1.75 × 10⁻⁵-8.75 × 10⁻⁴ 8.75 ×10⁻⁶ 0.999 31 Capillary 1.0 × 10⁻⁷-2.0 × 10⁻⁴  5.0 × 10⁻⁸ 0.9995 32Electrophoresis DPV 1.0 × 10⁻⁷-2.0 × 10⁻⁵  2.0 × 10⁻⁸ 0.998 33 HPLC 0.2× 10⁻⁶-2.0 × 10⁻⁶  0.18 × 10-06 0.9975 34 SWV  6.0 × 10⁻⁶-240 × 10⁻⁶1.98 × 10⁻⁶ 0.9996 35

Application of the proposed method for the determination of MTZ in realsamples. Determination of MTZ in tablet samples was examined todemonstrate the ability of the SERS method for the determination of MTZin real samples. The proposed method was applied for the determinationof MTZ in the commercial pharmaceutical dosage forms, tablet samples. Inorder to access the matrix effect, the relative recoveries of the methodwere calculated. The obtained results, shown in Table 5, indicate theaccuracy of the method, as well as the low interference limits caused bythe frequently encountered excipients and the degradation products.Thus, the SERS method retained its efficiency for the determination ofMTZ in real samples.

TABLE 5 Determination of MTZ in pharmaceutical tablet samples (n = 3);Recovered concentrations obtained for MTZ using a SERS method withG-D-Ag and calibration curve at 1369 cm⁻¹ (n = 3). Confidence SampleExpected Found Recovery % interval Bias (%) Tablet 1 5 mg/g 4.93 mg/g98.6 0.31 × 10⁻⁶M −1.4 Tablet 2 5 mg/g 4.88 mg/g 97.6 0.31 × 10⁻⁶M −2.4Spiked 1 2.5 × 10⁻⁶M 2.61 × 10⁻⁶M 104.4 0.48 × 10⁻⁶M +4.4 Spiked 3 5.0 ×10⁻⁶M 5.13 × 10⁻⁶M 102.6 0.72 × 10⁻⁶M +2.6

As shown herein, the inventors have synthesized graphene functionalizedwith polyamidoamine dendrimer decorated with silver nanoparticles(G-D-Ag) and characterized it by using various techniques including SEM,TEM, FTIR and UV.

The SERS method was exploited to record the vibrational frequencies ofMTZ adsorbed on G-D-Ag. The optimized conformation and vibrationalassignments of MTZ were carried out using a DFT calculation with aB3LYP/6-311++G (d, p) basis set. The vibration assignments and the wavenumber of vibration frequency bands in the theoretical spectra were inagreement with those of the experimental spectra. Most of the bandsrelated to N and S atom were apparently enhanced and slightly shifted.These results confirm that MTZ molecules were adsorbed on the G-D-Ag,probably through the lone pair on the N and S atoms. The correlationbetween the logarithmical scale of MTZ concentration and the SERS signalwas linear within a dynamic range of 10⁻⁶±10⁻¹¹ and R² of 0.9976, andwith good detection limits down to 1.43×10⁻¹² (or any intermediate valueor subrange). This detection limit was calculated as three-times thebaseline noise. The experimental detection limit was 1×10⁻¹¹M.

Terminology. Terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The headings (as “Background” and “Summary” and sub-headings used hereinare intended only for general organization of topics within the presentinvention, and are not intended to limit the disclosure of the presentinvention or any aspect thereof. In particular, subject matter disclosedin the “Background” may include novel technology and may not constitutea recitation of prior art. Subject matter disclosed in the “Summary” isnot an exhaustive or complete disclosure of the entire scope of thetechnology or any embodiments thereof. Classification or discussion of amaterial within a section of this specification as having a particularutility is made for convenience, and no inference should be drawn thatthe material must necessarily or solely function in accordance with itsclassification herein when it is used in any given composition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper”, “in front of” or “behind” and the like, may be used herein forease of description to describe one element or feature's relationship toanother element(s) or feature(s) as illustrated in the figures. It willbe understood that the spatially relative terms are intended toencompass different orientations of the device in use or operation inaddition to the orientation depicted in the figures. For example, if adevice in the figures is inverted, elements described as “under” or“beneath” other elements or features would then be oriented “over” theother elements or features. Thus, the exemplary term “under” canencompass both an orientation of over and under. The device may beotherwise oriented (rotated 90 degrees or at other orientations) and thespatially relative descriptors used herein interpreted accordingly.Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal”and the like are used herein for the purpose of explanation only unlessspecifically indicated otherwise.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A methimazole adsorbant sample slide,comprising: a glass substrate, and an adsorbant coating on a surface ofthe glass substrate, wherein the adsorbant coating containsgraphene-dendrimer-stabilized silver nanoparticles (G-D-Ag), wherein theG-D-Ag of the adsorbant coating comprise: a graphene oxide sheetchemically bonded with at least 2 polyamidoamine (“PAMAM”) dendrimersthrough carboxamide bonds as represented by formula (I):

wherein X is -A-B—NH₂, -A-B—N-(A-B—NH₂)₂, or -A-B—N-[A-B—N-(A-B—NH₂)₂]₂;A is —CH₂CH₂C(O)—; B is —NHCH₂CH₂—; Graphene represents the grapheneoxide sheet; and m is a positive integer in the range of 2-100; andsilver nanoparticles bound to the graphene oxide sheet supportedpolyamidoamine (“PAMAM”) dendrimer; wherein a weight ratio of the silvernanoparticles relative to the graphene oxide sheet is in the range of1:1 to 3:1.
 2. The methimazole adsorbant sample slide of claim 1,wherein the silver nanoparticles of the G-D-Ag in the adsorbant coatinghave a mean diameter of no more than 37 nm.
 3. The methimazole adsorbantsample slide of claim 1, wherein the silver nanoparticles of the G-D-Agin the adsorbant coating have a mean diameter of no more than 18 nm. 4.The methimazole adsorbant sample slide of claim 1, comprising silicaglass coated with at least one layer of the G-D-Ag.
 5. The methimazoleadsorbant sample slide of claim 1, wherein the adsorbant coating rangesin thickness from 10 nm to 100 μm.
 6. A method for detecting methimazole(“MTZ”) or determining a concentration of methimazole in a samplecomprising contacting the sample containing or suspected of containingMTZ with the methimazole adsorbant sample slide of claim 1, adsorbingthe MTZ to the G-D-Ag of the adsorbant coating, and performingsurface-enhanced Raman scattering (SERS) of the G-D-Ag adsorbed MTZ. 7.The method of claim 6, wherein the sample is a pharmaceutical, drug orchemical sample and not a biological sample from a subject.
 8. Themethod of claim 6, wherein the sample is serum, plasma, urine or otherbiological sample.
 9. The method of claim 6, wherein the sample is humanserum, plasma, urine or other human biological sample.
 10. The method ofclaim 6, wherein the sample is obtained from Felis catus (domestic cat)or other member of the family Felidae.
 11. The method of claim 6,wherein the sample is from a subject having hyperthyroidism or at riskthereof.
 12. The method of claim 6, wherein the sample is from a femalewho is pregnant or who may become pregnant.
 13. The method of claim 6,wherein the sample is from a subject having, genetically predisposed tohaving, or at risk of having agranulocytosis, aplastic anemia(pancytopenia), ANCA-positive vasculitis, exfoliative dermatitis,hepatitis, or hepatic dysfunction or at least one symptom thereof; orwherein the sample is from a subject having anorexia, pruritus, or rightupper quadrant pain or other symptom of hepatic dysfunction or whereinthe sample is from a subject having an abnormal liver function test.